stage of cell entry. Virus particles with such amino acid alterations can bind to target cells and convert to A particles but are blocked at a subsequent, unidentified step. During poliovirus assembly, VP4 and VP2 are part of the precursor VP0, which remains uncleaved until the viral RNA has been encapsidated. The cleavage of VP0 during poliovirus assembly therefore primes the capsid for uncoating by separating VP4 from VP2.
In cells in culture, release of the poliovirus genome occurs from within early endosomes located close (within 100 to 200 nm) to the plasma membrane (Fig. 5.22). Uncoating is dependent on actin and tyrosine kinases, possibly for movement of the capsid via the network of actin filaments. Movement is not dependent on dynamin, clathrin, caveolin, or flotillin (a marker protein for clathrin- and caveolin-independent endocytosis); endosome acidification; or microtubules. The trigger for RNA release from early endosomes is not known but is clearly dependent on prior interaction with CD155. This conclusion derives from the finding that antibody-poliovirus complexes can bind to cells that produce Fc receptors but cannot infect them. As the Fc receptor is known to be endocytosed, these results suggest that interaction of poliovirus with CD155 is required to induce the conformational changes in the particle that are necessary for uncoating.
Figure 5.21 Stepwise uncoating of adenovirus. (A) Adenovirus fiber proteins bind a primary cell receptor, often CAR (Coxsackievirus and adenovirus receptor). Subsequently, interaction of the penton base with vibronectin-binding integrins αvβ3 and αvβ5 leads to internalization by endocytosis. Fibers are released from the capsid during uptake. The capsid protein is further destabilized in the endosome, likely triggered by low pH, and releases several viral proteins including protein VI (yellow). The hydrophobic N terminus of protein VI disrupts the endosome membrane, leading to release of the subviral particle into the cytoplasm. This particle is transported in the cytoplasm along microtubules and docks onto the nuclear pore complex, where further disassembly occurs to release the viral DNA into the nucleus. Individual steps in entry have been timed, and the overall process from receptor binding to nuclear entry takes a total 85 to 105 minutes. Data from Greber UF et al. 1993. Cell 75:477–486, 1993; and Trotman LC et al. 2001. Nat Cell Biol 3:1092–1100. (B) Electron micrograph of adenovirus type 2 particles bound to a microtubule (top) and bound to the cytoplasmic face of the nuclear pore complex (bottom). Reprinted from Greber UF et al. 1994. Trends Microbiol 2:52–56, with permission. Courtesy of Ari Helenius, Urs Greber, and Paul Webster, University of Zurich.
A critical regulator of the receptor-induced structural transitions of poliovirus particles appears to be a hydrophobic tunnel located below the surface of each structural unit (Fig. 5.22). The tunnel opens at the base of the canyon and extends toward the 5-fold axis of symmetry. In poliovirus type 1, each tunnel is occupied by a molecule of sphingosine. Similar lipids have been observed in the capsids of other picornaviruses. Because of the symmetry of the capsid, each virus particle may contain up to 60 lipid molecules. These lipids are thought to contribute to the stability of the native virus particle by locking the capsid in a stable conformation. Consequently, removal of the lipid is probably necessary to endow the particle with sufficient flexibility to permit the RNA to leave the protein shell.
The viral genome is released from the endosome, and it is usually assumed that the 5′ end of (+) strand RNAs is the first to leave the capsid, to allow immediate initiation of translation by ribosomes. This assumption is incorrect for rhinovirus type 2: exit of viral RNA starts from the 3′ end. This directionality is a consequence of how the viral RNA is packaged in the virus particle, with the 3′ end near the location of pore formation in the altered particle. Whether such directionality is a general feature of nonenveloped (+) strand RNA viruses is unknown.
Similar to picornaviruses, another family of nonenveloped (+) strand RNA viruses, caliciviruses, also form pores in the endosomal membrane. Binding to the receptor triggers conformational changes in the viral capsid, and following endocytosis, the capsid protein VP2 forms a large portal at the 3-fold axis of symmetry. This portal would allow delivery of the RNA genome to the cytoplasm.
Figure 5.22 Model for poliovirus entry into cells. The native virus particle (160S) binds to its cell receptor, CD155, and undergoes a receptor-mediated conformational transition resulting in the formation of altered (A) particles. Shortly after endocytosis and close to the plasma membrane, the viral RNA leaves the capsid. A long, umbilical connector is formed between the particles and the endosomal membrane that allows the RNA to escape. (Inset) Cross-section of poliovirus particle bound to CD155. Capsid pockets are occupied by lipids that may contribute to capsid stability.
Disrupting the Lysosomal Membrane
Most virus particles that enter cells by receptor-mediated endocytosis leave the pathway before the vesicles reach the lysosomal compartment. This departure is not surprising, for lysosomes contain proteases and nucleases that would degrade virus particles. However, these enzymes play an important role during the uncoating of members of the Reoviridae.
Orthoreoviruses are naked icosahedral viruses containing a double-stranded RNA genome of 10 segments. The viral capsid is a double-shelled structure assembled from eight different proteins. These virus particles bind to cell receptors via protein σ1 and are internalized into cells by endocytosis (Fig. 5.23). The intact virus particle comprises two concentric, icosahedrally organized protein capsids. The outer capsid is made up largely of σ3 and μ1. The dense core shell is formed mainly by λ1 and σ2.
Infection of cells by reoviruses is sensitive to bafilomycin A1, an inhibitor of the endosomal proton pump, indicating that acidification is required for entry. Disassembly occurs in multiple steps while the virus particle travels within endosomes to the lysosome (Fig. 5.23A). The process is initiated with the acid-induced proteolysis that releases the 600 σ3 subunits of the capsid. The μ1 protein changes from a compact form to an extended flexible fiber, producing an infectious subviral particle (ISVP). The μ1 protein undergoes significant conformational changes and is cleaved at three sites, one of which releases the myristoylated N terminus, μ1N, which can insert into membranes (Fig. 5.23B). Both μ1N and μ1C are required for membrane penetration. Isolated ISVPs cause cell membranes to become permeable to toxins and produce pores in artificial membranes. These can also initiate an infection by penetrating the plasma membrane, entering the cytoplasm directly. Their infectivity is not sensitive to bafilomycin A1, further supporting the idea that these particles are primed for membrane entry and do not require further acidification for this process.
The core particles generated from infectious subviral particles after penetration into the cytoplasm adopt a third morphology and carry out viral mRNA synthesis. The core is produced by the release of 12 σ1 fibers and 600 μ1 subunits. In the transition from ISVP to core, domains of λ2 rotate upward and outward to form a turret-like structure (Fig. 5.23A).
